Available treatments for schizophrenia are not effective in treatment of all major symptoms associated with the disease and induce a number of dose-limiting adverse effects. Thus, there is a critical need to develop novel therapeutic agents for treatment of schizophrenia that have broader efficacy and fewer adverse effects. During the initial funding period of the Vanderbilt NCDDG for discovery of novel treatments for schizophrenia, we made tremendous progress in discovery of drug candidates for novel targets and partnering with pharmaceutical companies to advance these agents to clinical development. In addition, we built on previous clinical studies suggesting that selective activators of the M1 muscarinic acetylcholine receptor have potential for treatment of cognitive disturbances and negative symptoms associated with schizophrenia. We optimized novel highly selective M1 positive allosteric modulators (PAMs) and have extensively evaluated these compounds in animal models of cognitive function and in pre-GLP safety studies. We have identified VU0467319 as a highly optimized M1 PAM that possesses an excellent balance of properties required for selection as a clinical development candidate. We now propose continued support of the Vanderbilt NCDDG to advance VU0467319 into first in human studies and to rigorously establish the doses required to achieve activation of M1 in healthy human volunteers. Project 1 will develop and validate a new translational biomarker for M1 PAM activity that can be used as a surrogate for target engagement and dose-finding studies in early clinical development. Project 2 will provide scale-up synthesis of VU0467319 and other compounds and provide backup clinical candidates to respond to findings with VU0467319 during GLP safety assessment. Project 3 will focus on IND-enabling studies to open an IND to allow clinical evaluation of VU0467319. In Project 4, we will perform safety assessments of VU0467319 in healthy human volunteers and employ the biomarker developed in project 1 to establish the doses required to achieve adequate CNS activity in humans. In addition, project 4 will provide a preliminary assessment of effects of VU0467319 on cognitive function in humans. These studies will provide a fully validated clinical research tool for future clinical studies to fully evaluate the efficacy of a highly optimized M1 PAM in schizophrenia patients.